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Publications (4)15.72 Total impact

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    ABSTRACT: Class II fructose 1,6-bisphosphate aldolases (FBA; E.C. 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff-base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs has been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies on class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation/deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI/DHAP bound form of the enzyme and determined the X-ray structure of MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information plus site-directed mutagenesis and kinetic studies conducted on a series of residues within the active-site loop revealed that E169 facilitates a water mediated deprotonation/protonation step of the MtFBA reaction mechanism. Also, secondary isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.
    Biochemistry 01/2013; · 3.38 Impact Factor
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    ABSTRACT: Specific nascent peptides in the ribosome exit tunnel can elicit translation arrest. Such ribosome stalling is used for regulation of expression of some bacterial and eukaryotic genes. The stalling is sensitive to additional cellular cues, most commonly the binding of specific small-molecular-weight cofactors to the ribosome. The role of cofactors in programmed translation arrest is unknown. By analyzing nascent peptide- and antibiotic-dependent ribosome stalling that controls inducible expression of antibiotic resistance genes in bacteria, we have found that the antibiotic is directly recognized as a part of the translation modulating signal. Even minute structural alterations preclude it from assisting in ribosome stalling, indicating the importance of precise molecular interactions of the drug with the ribosome. One of the sensors that monitor the structure of the antibiotic is the 23S rRNA residue C2610, whose mutation reduces the efficiency of nascent peptide- and antibiotic-dependent ribosome stalling. These findings establish a new paradigm of the role of the cofactor in programmed translation arrest in which a small molecule is recognized along with specific nascent peptide sequences as a composite structure that provokes arrest of translation. A similar mechanism could be used by the ribosome to sense a variety of cellular metabolites.
    Proceedings of the National Academy of Sciences 06/2011; 108(26):10496-501. · 9.81 Impact Factor
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    ABSTRACT: Existing 14, 15 and 16-membered macrolide antibiotics, while effective for other bacterial infections, including some mycobacteria, have not demonstrated significant efficacy in tuberculosis. Therefore an attempt was made to optimize this class for activity against Mycobacterium tuberculosis through semisyntheses and bioassay. Approximately 300 macrolides were synthesized and screened for anti-TB activity. Structural modifications on erythromycin were carried out at positions 3, 6, 9, 11, and 12 of the 14-membered lactone ring; as well as at position 4'' of cladinose and position 2' of desosamine. In general, the synthesized macrolides belong to four subclasses: 9-oxime, 11,12-carbamate, 11,12-carbazate, and 6-O-substituted derivatives. Selected compounds were assessed for mammalian cell toxicity and in some cases were further assessed for CYP3A4 inhibition, microsome stability, in vivo tolerance and efficacy. The activity of 11,12-carbamates and carbazates as well as 9-oximes is highly influenced by the nature of the substitution at these positions. For hydrophilic macrolides, lipophilic substitution may result in enhanced potency, presumably by enhanced passive permeation through the cell envelope. This strategy, however, has limitations. Removal of the C-3 cladinose generally reduces the activity. Acetylation at C-2' or 4'' maintains potency of C-9 oximes but dramatically decreases that of 11,12-substituted compounds. Further significant increases in the potency of macrolides for M. tuberculosis may require a strategy for the concurrent reduction of ribosome methylation.
    Tuberculosis (Edinburgh, Scotland) 08/2008; 88 Suppl 1:S49-63. · 2.54 Impact Factor
  • Tuberculosis. 01/2008; 88.